Cytotoxic factors for modulating cell death

ABSTRACT

Cytotoxic factors having use in modulating cell death, and their use in methods of treating necrosis or apoptosis-related conditions are disclosed. The invention also relates to methods for identifying active agents useful in treating conditions related to cell death. The present inventors have found that different pathogens produce different cytotoxic factor(s) having anticancer activity. The substantially pure cytotoxic factors can be used in a method of treating an infectious disease or a cancer.

RELATED APPLICATIONS

The present application is a Continuation and claims the benefit, under35 U.S.C. § 120, of U.S. patent application Ser. No. 11/485,252, filedJul. 13, 2006, which is a Continuation of U.S. patent application Ser.No. 10/047,710, filed Jan. 15, 2002, and the benefit, under 35 U.S.C. §119, of U.S. Provisional Application Ser. No. 60/269,133, filed Feb. 15,2001, which is expressly incorporated fully herein by reference.

STATEMENT OF GOVERNMENTAL INTEREST

The subject matter of this application has been supported by researchgrants from the National Institutes of Health (NIH), Bethesda, Md.,U.S.A., (Grant Numbers AI 16790-21, ES 04050-16, AI 45541,, CA09432 andN01-CM97567). The government may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to cytotoxic factors secreted bypathogenic microorganisms and inhibitors of cytotoxic factors and theiruse thereof in modulating cell death by both necrosis and apoptosis. Thepresent invention also relates to methods of producing, isolating andidentifying cytotoxic factors useful in modulating apoptosis, and tocompositions incorporating substantially pure cytotoxic factors usefulin modulating cell death. The invention also relates to methods oftreating apoptosis-related conditions. More particularly, the inventionrelates to the use of a substantially pure cytotoxic factor in a methodof inducing apoptosis in a cancer cell and to the use of inhibitors ofthe cytotoxic factors in a method of treating an infection or otherpathogen-induced condition.

BACKGROUND

Infectious diseases can be a product of a number of environmentalfactors. Underlying any infectious disease is a causative infectiousagent. The infectious agent typically is a pathogenic microorganism, forexample, a pathogenic bacterium. The degree or ability of a pathogenicmicroorganism to overcome defense mechanisms and cause a disease isrelated to its virulence. Pathogenic microorganisms are known to expresscytotoxic factors, which allow the pathogen to defend itself from thehost immune system and prevent phagocytes (e.g., macrophages and mastcells) from eliminating the pathogen from the body. When the pathogenicmicroorganisms survive, the microorganisms can invade the host tissuesand proliferate, causing severe disease symptoms. Pathogenic bacteriahave been identified as a root cause of a variety of debilitating orfatal diseases including, for example, tuberculosis, cholera, whoopingcough, plague, and the like. To treat such severe infections, drugs, forexample, antibiotics, are administered that either kill the infectiousagent or disarm the cytotoxic factors so that the infectious agent is nolonger able to defend itself against the host immune system. However,pathogenic bacteria commonly develop resistance to antibiotics andimproved agents are needed to prevent the spread of infections due tosuch microorganisms.

A cancer is a malignant tumor of potentially unlimited growth. It isprimarily the pathogenic replication (a loss of normal regulatorycontrol) of various types of cells found in the human body. Initialtreatment of the disease is often surgery, radiation treatment or thecombination, but locally recurrent and metastatic disease is frequent.Chemotherapeutic treatments for some cancers are available but theseseldom induce long term regression. Hence they are not usually curative.Commonly, tumors and their metastases become refractory to chemotherapy,in an event known as the development of multidrug resistance. In manycases, tumors are inherently resistant to some classes ofchemotherapeutic agents. In addition, such treatments threatennoncancerous cells, are stressful to the human body, and produce manyside effects. Hence, improved agents are needed to prevent the spread ofcancer cells. It has been known that many cancers regress when patientsare infected with pathogenic bacteria. However, very little is knownabout how bacterial infections may cause regression of human, cancers.

SUMMARY

The present invention relates to cytotoxic factors that stimulate celldeath by necrosis or apoptosis. In one aspect, substantially purecytotoxic factors have been characterized and isolated. Substantiallypure cytotoxic factors are obtained by column, chromatographicfractionation of a growth medium which has been exposed to a pathogenicmicroorganism. Preferably, the production and secretion of suchcytotoxic factors are stimulated during growth of pathogenic organismsin the presence of mammalian proteins.

In another aspect of the present invention, the identification ofreceptors for mammalian proteins as a means of delineating virulent andavirulent microorganisms can lead to improved specificity for diseasetreatment.

Yet another aspect of the present invention relates to a method oftreating a condition related to cell death resistance or susceptibilitycomprising the step of administering a cytotoxic factor, an inhibitor ofa cytotoxic factor, or a variant or derivative thereof, optionallyincorporated in a pharmaceutical carrier.

The cytotoxic factor, or a variant or derivative thereof, can beincorporated into a pharmaceutical composition for use in the preventionand treatment of conditions related to abnormal cell proliferation. Forexample, a cytotoxic factor can be used to treat a cancer.

An inhibitor of a cytotoxic factor, or a variant or derivative thereof,can be used to treat a bacterial infection by preventing phagocytic celldeath and hence allowing the host immune system to combat an invadingpathogen.

In another embodiment of the present invention, cytotoxic factors, aswell as components of their secretion machinery, can be used ascandidates for vaccines against infectious agents.

The present invention also relates to a method of modulating cell deathcomprising the step of controlling secretion of cytotoxic factors. In apreferred embodiment, the cytotoxic factors can be used as anti-canceragents against a host of human cancer cells. In addition, cytotoxicfactors can be used as targets for drug development through screening orrational design of inhibitors.

These and other aspects, advantages, and features of the invention willbecome apparent from the following figures and detailed description ofthe preferred embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Chart showing the effect of 1.0 mM ATP on macrophage killing inabsence or in presence of the filtered growth, medium supernatant (SUP)or the hydroxyapatite flow through (HAFT). ATP-agarose flow through(AAFT) and Q-sepharose flow through (QSFT) column chromatographicfractions derived from B. cepacia growth medium. The extent ofmacrophage cell death is measured by release of the intracellular enzymelactate dehydrogenase (LDH). 2 μg of protein from each fraction was usedin the assay. All assays were carried out in triplicate and error barsare indicated.

FIG. 2. Chart showing the effect of filtered growth medium supernatant(SUP) and column chromatographic fractions (HAFT, AAFT and QSFT) of B.cepacia on macrophage cell death in the absence of ATP. The extent ofmacrophage cell death is measured by the release of the intracellularenzyme lactate dehydrogenase (LDH). All assays were carried out intriplicate and error bars are indicated.

FIG. 3. Graphs showing caspase activities (FIG. 3A-caspase-3; FIG.3B-caspase-9) in the cytosolic extracts of J774 macrophages treated withB. cepacia QSFT fraction. Cytosolic extracts were prepared frommacrophages incubated overnight with B. cepacia QSFT traction (10 μgprotein) and from untreated macrophages. The substrate for thedetermination of caspase-3 activity was Ac-DEVD-pNA(N-acetyl-Asp-Glu-Val-Asp-p-NO₂-aniline (SEQ ID NO: 1)). The substratefor caspase-9 activity was Ac-LEHD-pNA(N-acetyl-Leu-Glu-His-Asp-p-NO₂-aniline (SEQ ID NO: 2)). Extracts wereincubated with the substrate at 37° C. for the times indicated. 10 μg ofmacrophage cytosolic protein was used in each case. Release of pNA(p-nitroaniline) was determined spectrophotometrically at 405 nm.

FIG. 4. Chart showing cytotoxicity, as measured by % lactatedehydrogenase (LDH) release, in macrophages in presence of azurin (Az),cytochrome c₅₅₁ (Cyt C₅₅₁) and combination thereof. The numbersrepresent μg protein. The buffer control (buffer) is shown at right.

FIG. 5. Chart showing the effects of anti-azurin and anti-cytochromec551 antibodies on cytotoxicity of B. cepacia (A) and M. bovis (B) QSFTfractions and in the presence of preimmune serum. A, azurin (50 μg); C,cytochrome c551 (25 μg); ab, combination of anti-azurin andanti-cytochrome c551 antibodies; P, preimmune serum. 2 μg of QSFTfraction were used in each assay. The numbers after ab and P representμg of the antibody or preimmune protein. Results shown aremeans±standard deviations of triplicate experiments.

FIG. 6. Graph showing the effect of post injection of azurin/cytochromec₅₅₁ in nude mice on the size of the tumor after induction of melanomatumor cells (UISO-Mel-2). Approximately 10⁶ UISO-Mel-2 cells wereinjected subcutaneously in nude mice followed by once weeklyintraperitoneal injections of either citrate buffer (control), a knownanti-melanoma drug DTIC (7.5 μg) or three times per week a high (150 μgazurin/75 μg cytochrome c₅₅₁) or low (10 μg azurin/5 μg cytochrome c₅₅₁)dose of azurin/cytochrome c₅₅₁ mixture for 4 weeks. At various times,the sizes (tumor volume) of the tumors in control (buffer treated),DTIC-treated and high and low dose azurin/cytochrome c₅₅₁-treated micewere determined and plotted graphically.

FIG. 7. Graph showing gain or loss of weight of the mice during theexperiment described under FIG. 6. During the course of the aboveexperiment, the mice were weighed on a scale and the weights in gramsnoted.

FIG. 8, Graph showing regression of Mel-6 tumor in nude mice treatedwith M. Bovis QSFT fraction in the presence or absence of azurin (AZ).Approximately 10⁶ UISO-Mel-6 cells were injected subcutaneously in nudemice. Small tumors developed after approximately one week. The mice werethen intraperitonealy injected with phosphate buffered saline (control),M. Bovis QSFT fraction or a mixture of M. Bovis QSFT fraction andazurin.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions.

For the purposes of the description herein, the term “cytotoxic factor”refers to a factor secreted by a pathogenic microorganism and whichstimulates cell death by necrosis or apoptosis. The term“ATP-dependent”, when used to modify the term “cytotoxic factor” refersto a cytotoxic factor which acts to cause cell death in the presence ofadenosine 5′-triphosphate (ATP). The term “ATP-independent”, when usedto modify the term “cytotoxic factor” refers to a cytotoxic factor whichacts to cause cell death in the absence of ATP.

For the purposes of the description herein, the term “treatment”includes preventing, lowering, stopping, or reversing the progression orseverity of the condition or symptoms being treated. As such, the term“treatment” includes both medical, therapeutic, and/or prophylacticadministration, as appropriate.

As used herein, the term “a condition related to resistance to celldeath” refers to a disease, state, or ailment characterized by at leasta tendency for prolonged cell life when compared with a healthy cell oflike kind as determined by a reasonable, skilled physician or clinician.The term “a condition related to cell death susceptibility”, as usedherein, refers to a disease, state, or ailment characterized by at leasta tendency for premature cell death when compared with a healthy cell oflike kind as determined by a reasonable, skilled physician or clinician.

The term “substantially pure”, when used to modify the term “cytotoxicfactor” or “virulence factor”, as used herein, refers to a factorisolated from the secreted growth medium in a form substantially freeof, or unadulterated by, active inhibitory compounds. The term“substantially pure” refers to a factor in an amount of at least about75%, by weight, of isolated fraction, or at least “75% substantiallypure”. More preferably, the term “substantially pure” refers to acompound of at least about 85%, by weight, active compound, or at least“95% substantially pure”. The substantially pure cytotoxic factor orvirulence factor can be used in combination with one or more othersubstantially pure compounds or isolated cytotoxic factors.

As used herein, the term “a variant or derivative thereof” refers to acompound or substance obtained by chemical modification or manipulationof genes encoding the compound or substance. When referring to a variantor derivative of a cytotoxic factor, the variant or derivative can beobtained by chemical modification of the cytotoxic factor, or bymanipulation of genes encoding such cytotoxic factors, for example byaltering the basic composition or characteristics of the cytotoxicfactor, but not its toxicity. Similarly, a derivative of an inhibitor ofa cytotoxic factor can include chemical modifications to the chemicalstructure of the inhibitor or manipulation of genes encoding theinhibitor. For example, the antibiotic penicillin can be chemicallymodified to provide derivatives that are more potent or have a widerspectrum than penicillin itself.

A “therapeutically effective amount” is an amount effective to preventdevelopment of, or to alleviate the existing symptoms of, the subjectbeing treated. Determination of a therapeutically effective amount iswell within the capability of those skilled in the art.

General.

The present invention provides cytotoxic (or virulence) factors that aresecreted by pathogenic microorganisms and that stimulate cell death bynecrosis or apoptosis. When pathogenic microorganisms invade human oranimal tissues, phagocytic cells are a first line of defense in the hostimmune system. Typically, phagocytes seek out and destroy foreignpathogens invading the body. However, cytotoxic factors secreted bymicrobial pathogens can stimulate cell death in the phagocytic cells.Thus, the phagocytes are prevented from performing their protectiveimmune function.

The inventors have previously reported that many pathogenic bacteriasecrete ATP-dependent cytotoxic factors, for example ATP-utilizingenzymes, that cause phagocytic cell death by necrosis. [Zaborina O. etal., Infect. Immun. 67: 5231-5242 (1999); Melnikov A. et al, Mol.Microbiol. 36: 1481-1493 (2000); and Punj et al. Infect. Immun. 68:4930-4937 (2000).] ATP-utilizing enzymes act on various energy-relatednucleotide derivatives such as ATP, adenosine 5′-diphosphate (ADP),adenosine 5′-monophosphate (AMP), or adenosine, converting them tovarious products that in turn can modulate the death of phagocytic cellssuch as macrophages and mast cells through activation of purinergicreceptors.

One aspect of the present invention relates to the discovery thatATP-independent cytotoxic factors, for example redox proteins, are alsosecreted by some species of pathogenic microorganisms, and that suchfactors cause phagocytic cell death by apoptosis. [see Zaborina O. etal. Microbiology 146: 2521-2530 (2000).]

Another aspect of the present invention relates to the surprisingdiscovery that ATP-Independent cytotoxic factors induce apoptosis incancer cells. Normally cancer cells are not susceptible to apoptoticdeath. It is known that mammalian cell apoptosis requires the presenceof p53 protein. However, in 50% of human cancers, inactivating mutationsin the gene encoding the p53 tumor suppressor protein are present.Although it is also known that p53 regulates the expression of redoxproteins in mammalian cells, mammalian redox proteins have not beendirectly implicated in cancer cell apoptosis. Neither has the role ofmicrobial ATP-independent cytotoxic factors in inducing apoptosis incancer cells or in reducing tumor size been shown. Thus, such cytotoxicfactors may be used to treat a condition related to resistance to celldeath. Such conditions may include, for example, human melanoma,leukemia, breast cancer, ovarian cancer, lung cancer, mesenchymalcancer, colon cancer and aerodigestive tract cancers (e.g. stomach,esophagus, larynx and oral cancers).

Another aspect of the present invention relates to methods ofidentification and characterization of cytotoxic factors secreted bypathogenic microorganisms. Such methods can provide a means fordiscovering appropriate inhibitors or stimulators of cell death.Inhibitors and stimulators can be developed as pharmaceutical drugs andused to treat conditions characterized by resistance or susceptibilityto cell death.

Another aspect of the invention relates to cytotoxic factors that havebeen characterized and isolated and to inhibitors of such cytotoxicfactors. The cytotoxic factors can be activated or inactivated inaccordance with a method of the invention to prevent or treat acondition related to cell death. An inhibitor of a cytotoxic factor canbe used to treat a condition related to cell death susceptibility.

Secretion of Cytotoxic Factors.

In one aspect of the present invention, cytotoxic factors of the presentinvention are secreted by a number of different pathogenicmicroorganisms, including bacteria and protozoa. Examples of pathogenicbacteria suitable for providing the cytotoxic factors include, but arenot limited to, Pseudomonas aeruginosa, Burkholderia cepacia, Vibriocholerae, and Mycobacterium bovis. In addition, cytotoxic factors aresecreted by pathogens, such as Leishmania amazonemis and Brugia malayi.

P. aeruginosa, an opportunistic pathogen, B. cepacia, which causes fatalinfections in patients suffering from cystic fibrosis and chronicgranulomatous disease, Vibrio cholerae, the intestinal pathogen thatcauses cholera and the slow-growing virulent group of mycobacteria, suchas M. tuberculosis or M. bovis, that cause tuberculosis have been foundto secrete ATP-utilizing enzymes.

In addition to secreting ATP-utilizing enzymes, the inventors have foundthat P. aeruginosa secretes ATP-independent cytotoxic factors. Thesehave been identified as two redox proteins, azurin and cytochrome c₅₅₁ .B. cepacia has also been shown to secrete the redox proteins. M. bovishas been shown to also secrete cytotoxic factors having highATP-independent cytotoxicity towards phagocytic cells.

Stimulation of the Secretion of Cytotoxic Factors in the Presence ofMammalian Proteins.

In another aspect of the present invention, production and secretion ofcytotoxic factors are stimulated during growth of pathogenic organismsin the presence of mammalian proteins. For example, the secretion ofcytotoxic factors by pathogenic microorganisms such as P. aeruginosa, M.bovis and B. cepacia is stimulated by the presence of mammalian proteinssuch as kappa-casein, bovine serum albumin, ovalbumin orα2-macroglobulin. It is suggested, but not relied upon herein, that thepathogenic microorganisms sense the presence of certain mammalianproteins as indicative of the mammalian host environment, therebyopening up the secretion machinery for the cytotoxic agents to counterand subvert host defense.

The inventors have determined that several clinical (virulent) isolatesof B. cepacia secrete large amounts of ATP-utilizing enzymes such asadenylate kinase or 5′-nucleotidase, while several environmental(avirulent) isolates secreted only reduced amounts of these enzymes, inclinical isolates, such as B. cepacia strain 38, the level of secretionof cytotoxic factor is greatly enhanced in the presence ofα2-macroglobulin in the growth medium. The secreted products fromclinical isolates have a higher level of cytotoxicity towardsmacrophages and mast cells than that from environmental isolates. Theclinical isolates that demonstrate enhanced secretion of cytotoxicfactors in the presence of α2-macroglobulin also demonstrate thepresence of the receptors for α2-macroglobulin on their surface.

In a preferred embodiment of the present invention, the production andsecretion, of ATP-independent cytotoxic factors are stimulated duringgrowth of pathogenic organisms in the presence of mammalian proteins.

Hence, increased secretion of cytotoxic factors can be obtained bygrowing pathogenic organisms in growth media containing mammalianproteins. Suitable growth media are, for example, L broth, nutrientbroth, Trypticase soy broth and tryptone-yeast extract both (DifcoLaboratories, Maryland, U.S.A.). Typically, approximately 500 ml to1,000 ml of sterile autoclaved growth medium are inoculated with betweenabout 10⁴ to 10⁶ cells/ml. The inoculated medium is then incubated underconditions suitable to allow growth of the microorganism, usually on arotary shaker at 30° C. to 37° C. Selection of growth media, incubationconditions, and other factors allowing successful culture of bacteriaand other microorganisms will be clearly apparent to one skilled in theart. The inventors have observed that maximum concentrations ofcytotoxic factors in the growth medium occur late in the exponentialgrowth phase and early in the stationary growth phase.

In another embodiment of the present invention, the identification ofreceptors for mammalian proteins provides a means of delineatingvirulent and avirulent strains of microorganisms. For example, thepresence of the receptors for α2-macroglobulin primarily in clinicalisolates, but not in environmental isolates, not only correlates withthe ability of the former to secrete cytotoxic agents as weapons againstthe host defense, but also allows delineation between the clinical,virulent strains with the environmental, avirulent strains. Hence,virulent strains of organisms can be identified and then tested fortheir antibiotic sensitivity or for other clinical purposes.

Purification of ATP-Independent Cytotoxic Factors.

In another aspect of the present invention, substantially pureATP-independent cytotoxic factors are obtained by column chromatographicfractionation of the growth medium of the secreting microorganism.Preferably, the bacterial cells are removed from the growth medium priorto fractionation. This may be achieved by initial centrifugation andsubsequent filtering the growth medium. Suitable filters are typicallyless than or equal to about 0.5 μm pore size and preferably about 0.2μm. However, other methods of pathogen removal will be known to thoseskilled in the art.

Unfractionated growth media do not have high ATP-independent cytotoxicactivity and hence column chromatographic fractionation is necessary toenhance apoptosis-inducing activity. Fractionation removes ATP-dependentcytotoxic factors, ft is also suggested, but not relied upon herein,that fractionation also removes inhibitors of ATP-independent cytotoxicfactors that may be present in the unfractionated growth medium.

Chromatographic techniques useful in purifying cytotoxic factors will beknown to those skilled in the art. These include, for example,ion-exchange chromatography, hydroxyapatite chromatography, affinitychromatography, and gel-filtration chromatography. Chromatographiccolumns useful in the fractionation of bacterial growth media include,for example: Hydroxyapatite; Superdex 75 or 200; Superose 6 or 12;Sephacryl S; Sephadex G or Sephadex LH; Mono Q or Mono S; Q-Sepharose;DEAE Sepharose or CM Sepharose; Sepharose XL; ATP-Sepharose; Hi TrapBlue; Blue Sepharose; DNA Cellulose or Sepharose 2B, 4B or 6B, availablefrom Amersham Pharmacia Biotech AB, Uppsala, Sweden or Bio-RadLaboratories, Hercules, Calif., U.S.A.

Preferably, ATP-utilizing enzymes are isolated by column chromatographicfractionation as flow-through or eluted fractions of hydroxyapatite andATP-agarose columns. During such fractionation, the ATP-utilizingenzymes, such as ATPase or adenylate kinase are adsorbed on the columnand can be removed or purified further. (See, for example, Markaryan etal., J. Bacteriol., 183, pp 3345-3352, 2001.)

In a preferred embodiment of the present invention, ATP-independentcytotoxic factors are isolated as flow-through fractions of Q-sepharosecolumns (QSFT). Q-sepharose is a quaternary ammonium strong anionexchanger. Such columns can be obtained from Amersham Pharmacia BiotechAB, Uppsala, Sweden. The supernatant (SUP) or other column fractionssuch as hydroxyapatite column flow through fraction (HAFT) orATP-agarose column flow through fraction (AAFT) do not normally showhigh ATP-independent cytotoxicity.

Characterization of ATP-Independent Cytotoxic Factors.

In a further aspect of the present invention, fractionated growth mediaare tested to determine the presence of ATP-independent cytotoxicfactors. The extent of cell death may be measured by the release of theintracellular enzyme lactate dehydrogenase (LDH) as described inZaborina et al., Infection and Immunity. 67, 5231-5242 (1999) andZaborina et al. Microbiology, 146, 2521-2530 (2000).

The ability of ATP-independent cytotoxic factors to induce apoptosis maybe observed by mitosensor ApoAlert confocal microscopy using aMITOSENSOR™ APOLER™ Mitochondrial Membrane Sensor kit (ClontechLaboratories, Inc., Palo Alto, Calif., U.S.A.). In the assay, healthy,non-apoptotic cells fluoresce red while apoptotically dead cellsfluoresce green. A combination of red and green produces yellowfluorescing cells that represent apoptotically dying cells. See Zaborinaet al. Microbiology, 146, 2521-2530 (2000).

Apoptosis is mediated via activation of a cascade of enzymes known ascaspases, which are cysteine proteases cleaving at aspartic residues.Hence, apoptosis may also be detected by measuring two important caspaseactivities, namely that of caspase 9 and caspase-3, which are known tobe activated during apoptosis by the oligomerization of the cytochrome creleased from mitochondria with a cytosolic protein Apaf-1, using themethod described in Zou et al., J. Biol. Chem., 274: 11549-11556 (1999).

In addition, apoptosis may be observed by detecting apoptosis-inducednuclear DNA fragmentation using the APOLERT DNA fragmentation kit(Clontech Laboratories, Inc., Palo Alto, Calif., U.S.A.). This assay isbased on terminal deoxynuclotidykransferase (Tdt)—mediated dUTP nick-endlabeling (TUNEL), where Tdt catalyzes the incorporation offluorescein-dUTP at the free 3′-hydroxyl ends of fragmented DNA in cellsundergoing apoptosis. The incorporation of fluorescein-dUTP in thefragmented nuclear DNA generates green fluorescence which is detected byconfocal microscopy.

In a preferred embodiment of the present invention, fractionated growthmedia are tested to determine the ability of such fractions to induceapoptosis. Such methods are useful in the identification andcharacterization of ATP-independent cytotoxic factors.

Identification of ATP-Independent Cytotoxic Factors.

In another aspect, this invention provides characterized cytotoxicfactors exhibiting ATP-independent apoptosis-triggering cytotoxicity.The inventors have found that the QSFT fraction of P. aeruginosa and B.cepacia is enriched with two proteins, azurin and cytochrome c₅₅₁. Theidentification of these two proteins is based on their separation onSDS-PAGE and identification of their N-terminal amino acid sequences. Incontrast, SDS-PAGE analysis of the M. bovis QSFT fraction shows a thick65 kDa band of bovine serum albumin (BSA), which is a constituent of the7119 medium used for growing M. bovis, as well as several bands ofgreater than 45 kDa molecular mass, but not the bands characteristic ofcytochrome c₅₅₁ or azurin. (See Example 9.)

Azurin and/or cytochrome c₅₅₁ and the QSFT fractions exhibitapoptosis-triggering cytotoxicity towards phagocytic cells. A purifiedazurin; cytochrome c₅₅₁ mixture, or the B. cepacia QSFT fraction,treated with a mixture of anti-azurin and anti-cytochrome c₅₅₁antibodies, show greatly diminished macrophage cytotoxicity. Incontrast, the M. bovis QSFT fraction, when pretreated withanti-azurin/anti-cytochrome c₅₅₁ antibodies, shows very little reductionin cytotoxicity, confirming that M. bovis QSFT fraction containscytotoxic factors other than azurin or cytochrome c₅₅₁. Thus differentpathogens secrete different apoptosis-inducing cytotoxic factors, all ofwhich would be excellent targets for anti-infective drug development.

Induction of Apoptosis in Cancer Cells by ATP-Independent CytotoxicFactors.

The present invention provides methods of using ATP-independentcytotoxic factors to induce apoptotic cell death in cancer cells.ATP-independent cytotoxic factors, such as azurin and cytochrome c₅₅₁,can be used to treat conditions related to an abnormal failure of celldeath. It is well known that cancer cells are not prone to undergoingapoptosis. In accordance with one aspect of the present invention,administering a cytotoxic factor or active agent stimulating cytotoxicfactor secretion in an amount sufficient to induce cancer cell apoptosiswould be beneficial in reducing tumor size in vivo and retarding thegrowth of tumors. For example. Tests comparing azurin and cytochromec₅₅₁ to a known anti-melanoma cancer drug [5-(3,3′-N,N′-dimethyltriazen-1-yl)-imidazole-4-carhoxyamide] (DTIC) show that a mixture ofazurin and cytochrome C₅₅₁ provides a potent, non-toxic composition thatpromotes tumor regression in vivo in nude mice.

Use of Cytotoxic Factors in the Treatment of Infectious Disease.

In another aspect of the present invention, characterization ofcytotoxic factors can be useful for identifying new substances thatinhibit cell death, for example, in an infectious disease. For example,inhibition of the secretion or activity of an ATP-utilizing cytotoxicfactors, or the production of ATP, can reduce or eliminate cytotoxicactivity by a disease-causing pathogen.

Accordingly, appropriately administering a compound that inhibits thesecretion or activity of cytotoxic factors provides a useful tool foranti-infective development. Examples of active agents useful forinhibiting activity of cell death inducing cytotoxic factor can includeantibodies for cytotoxic factors, as well as analogues of ATP thatprevent the activation of ATP-utilizing enzymes. Examples of cytotoxicfactors and active agents for inhibiting or stimulating cytotoxic factorsecretion or expression include, but are not limited to, ATP-utilizingenzymes, redox proteins, activators of ATP-production, inhibitors of ATPproduction, activators of redox proteins, and inhibitors of redoxproteins.

Administration of Pharmaceutical Compositions Comprising CytotoxicFactors.

Pharmaceutical compositions comprising cytotoxic factors can bemanufactured in any conventional manner, e.g. by conventional mixing,dissolving, granulating, dragee-making, emulsifying, encapsulating,entrapping, or lyophilizing processes. The substantially pure cytotoxicfactor or other agent can be readily combined with a pharmaceuticallyacceptable carrier well-known in the art. Such carriers enable thepreparation to be formulated as a tablet, pill, dragee, capsule, liquid,gel, syrup, slurry, suspension, and the like. Suitable excipients canalso Include, for example, fillers and cellulose preparations. Otherexcipients can include, for example, flavoring agents, coloring agents,detackifiers, thickeners, and other acceptable additives, adjuvants, orbinders.

The compositions of the invention can be used in treatment of acondition related to cell death or in the prevention thereof. Thesubstantially pure cytotoxic factor can be administered in an amountsufficient to stimulate the natural response of the host immune systemand the secretion machinery of the host organism, for example as avaccine. Typically, the host organism is a mammal, such as a human oranimal.

The composition can be administered by any suitable route, for example,by oral, buccal, inhalation, sublingual, rectal, vaginal, transurethral,nasal, topical, percutaneous, i.e., transdermal or parenteral (includingintravenous, intramuscular, subcutaneous and intracoronary)administration. The compositions and pharmaceutical formulations thereofcan be administered in any amount effective to achieve its intendedpurpose. More specifically, the composition is administered in atherapeutically effective amount.

The exact formulation, route of administration, and dosage is determinedby the attending physician in view of the patient's condition. Dosageamount and interval can be adjusted individually to provide plasmalevels of the active cytotoxic factor which are sufficient to maintaintherapeutic effect. Generally, the desired cytotoxic factor isadministered in an admixture with a pharmaceutical carrier selected withregard to the intended route of administration and standardpharmaceutical practice. Pharmaceutical compositions used in accordancewith the present invention can be formulated in a conventional mannerusing one or more physiologically acceptable carriers comprisingexcipients and auxiliaries that facilitate processing of the cytotoxicfactor, active agents, for inhibiting or stimulating the secretion ofcytotoxic factors, or a mixture thereof into preparations which can beused therapeutically.

Stimulation and Inhibition of the Secretion of Cytotoxic Factors.

The identification and characterization of the cytotoxic factors alsocan lead to the development of methods of stimulating of cytotoxicfactor secretion. Pathogenic organisms have been shown to secrete largeamounts of cytotoxic factors in the presence of mammalian proteins. Thisprinciple can be modified in the human body to provide new methods ofstimulating desired, or inhibiting undesired, cytotoxic factorproduction. Such methods are useful for inhibiting or stimulating cellapoptosis. The understanding of the cytotoxic factors, and thecharacterization and development thereof, also allows for drugdevelopment and screening of active agents or compounds suitable formodulating the cytotoxic factor activity or secretion. The understandingof the secretion machinery related to cytotoxic factor secretion incells additionally provides new avenues of developing and identifyingthe design of useful inhibitors or stimulators of cytotoxic factors. Thedelineation and identification of the presence of receptors formammalian proteins also can be used as a means to differentiate betweenthe virulent and avirulent microorganisms, which can provide specificityin treating the disease. Components of the secretion machinery, as wellas cytotoxic factors themselves, can be used as vaccines.

Modification of Cytotoxic Factors.

Cytotoxic factors also can be chemically modified or genetically alteredto produce variants that lack an ATP-utilizing enzyme or redox activity,but retain toxicity. Mutations and/or truncations of the gene canproduce cytotoxic agents of varying compositions also demonstratingfunctional activity. In particular, truncated derivatives with highefficacy and low antigenicity can be produced from the originalcytotoxic factor. Such modified or altered cytotoxic factors, and suchcytotoxic agents, also are included in the scope of the presentinvention.

A more complete understanding of the present invention can be obtainedby reference to the following specific Examples. The Examples aredescribed solely for purposes of illustration and are not intended tolimit the scope of the invention. Changes in form and substitution ofequivalents are contemplated as circumstances may suggest or renderexpedient. Although specific terms have been employed herein, such termsare intended in a descriptive sense and not for purposes of limitations.Modifications and variations of the invention as hereinbefore set forthcan be made without departing from the spirit and scope thereof, and,therefore, only such limitations should be imposed as are indicated bythe appended claims.

EXAMPLES Example 1 Stimulation of the Secretion of Cytotoxic Factors byMammalian Proteins

Clinical and environmental isolates (five of each) of B. cepacia weregrown in proteose peptone-yeast extract (PPY) broth with and withoutadded α2-macroglobulin (1 mg/ml). After growth for 10 hours at 34° C. ona shaker, a portion of the growth medium from each culture wascentrifuged and the supernatant filtered through a 0.22 μm milliporefilter to remove whole cells and debri. The filtered supernatant wasthen tested for adenylate kinase activity as described in Melnikov A. etal., Mol. Microbiol., 36: 1481-1493 (2000). Adenylate kinase transfersthe terminal phosphate from [_(γ)-³²P]ATP to AMP giving rise to ADP. Theproducts of this reaction were then detected by thin-layerchromatography. Secretion of adenylate kinase was minimal when B.cepacia cells were grown in PPY broth. However, secretion from theclinical isolates, but not for the environmental isolates, wasstimulated in the presence of α2-macroglobulin.

Immunofluorescence microscopy with anti-α2-macroglobulin antibody showedthat the clinical isolates had receptors that bound α2-macroglobulinwhile the environmental isolates lacked such receptors. The clinical andenvironmental isolates of B. cepacia were grown in absence or inpresence of 1 mg/ml α2-macroglobulin in PPY broth for 1 hr. Extraneousα2-macroglobulin was removed by washing with phosphate-buffered saline.The cells were incubated for 2 hours with fluorescein isothiocyanate(FITC)-conjugated α2-macroglobulin antibodies, obtained by injectingrabbits with α2-macroglobulin. After washing with phosphate-bufferedsaline, the FITC conjugated antibody treated cells were fixed in 16%paraformaldehyde, coated on poly-L-lysine coated slides, and examined byconfocal microscopy. Only the clinical isolates that showed enhancedcytotoxic factor secretion in the presence of α2-macroglobulinfluoresced (green fluorescing cells), demonstrating the presence of thereceptors for α2-macroglobulin.

Example 2 ATP-dependent Macrophage Killing by Filtered Supernatant orColumn Chromatographic Fractions Derived from B. cepacia Growth Medium

A clinical strain of B. cepacia (strain 38—collection number 95828. D.G. Allison, University of Manchester Institute of Science andTechnology, Manchester, UK) was grown in TB broth (10 g of Bactotryptone, 3 g of Bacto beef extract per liter of water) at 34° C. on ashaker to an OD_(550nm) of 1.3. The growth medium was then centrifugedand the supernatant filtered through a 0.22 μm millipore filter toremove whole cells and debri. Macrophage cells were isolated from J774cell lines and grown in RPMI medium 1640 (GIBRO-BRL, Grand Island, N.Y.)as described by Zaborina O. et al, Infect. Immun. 61: 5231-5242 (1999).The filtered growth medium was added to hydroxyapatite, ATP-agarose, andQ-sepharose columns in sequence. The flow-through fraction from thehydroxyapatite column (HAFT) was fractionated on the ATP-agarose column(AAFT). The AAFT fraction was then fractionated on the Q-sepharosecolumn (QSFT).

10⁶ macrophages were added to wells in a 96 well plate and incubated fortwo hours in a CO₂ incubator for attachment. 2 μg of protein from thesupernatant or the flow-through fraction from each of the above columnswas added to the wells and the plates incubated for 4 hrs in thepresence or absence of 1.0 mM ATP. The extent of macrophage cell deathwas then measured by the release of the intracellular enzyme lactatedehydrogenase (LDH) as described by Zaborina O. et al. Infect. Immun.,67: 5231-5242 (1999). The extent of macrophage killing, in the presenceand in the absence of 1.0 mM ATP, by the filtered supernatant (SUP) andthe HAFT, AAFT and QSFT column fractions is shown in FIG. 1. All assayswere carried out in triplicate and error bars are indicated.

Example 3 ATP-independent Macrophage Killing by Filtered Supernatant orColumn Chromatographic Fractions Derived from B. cepacia Growth Medium

The supernatant (SUP) and column chromatographic fractions (HAFT, AAFTand QSFT) of B. cepacia growth medium were as in Example 2. Macrophageisolation was as in Example 2. The extent of macrophage cell death hasbeen determined by release of LDH as in Example 2 and is shown in FIG.2. Only the QSFT traction shows high ATP-independent cytotoxicitytowards macrophages.

Example 4 Induction of Apoptosis in Macrophages by P. aeruginosaCytotoxic Factor

P. aeruginosa was grown in L broth at 37° C. for 12 hours to anOD_(550nm) of 1.2. The growth medium was then centrifuged and thesupernatant filtered through a 0.22 μm filter. Supernatant (SUP) andcolumn chromatographic fractions (HAFT, AAFT and QSFT) were collected asin Example 2. Macrophage isolation was as in Example 2. 2 μg of proteinfrom the supernatant or one of the flow-through fractions was added to1×10⁵ macrophages in 200 μl of RPMI medium and the mixture incubatedovernight. Induction of apoptosis in macrophages either untreated ortreated by overnight incubation with the SUP or the HAFT, AAFT or QSFTfractions was measured by confocal microscopy using the ApoAlertMitochondria Membrane Sensor kit (Clontech Laboratories, Inc., PaloAlto, Calif., U.S.A.) as described by Zaborina O. et al., Microbiology146: 2521-2530 (2000).

In this assay, healthy, non-apoptotic cells fluoresce red whileapoptotically dead cells fluoresce green. A combination of red and greenproduces yellow fluorescing cells, indicating apoptotically dying cells.Nontreated macrophages or macrophages treated overnight with the SUP,HAFT or AAFT fractions fluoresced primarily red, indicating a lack ofapoptotic cell death. Macrophages treated overnight with the QSFTfraction fluoresced mostly green, indicating the apoptotic death of mostof the macrophages. A time course study showed that apoptosis set in atabout 6 hours (indicated by a combination of red and green fluorescencemaking the cell yellow) and was complete in 12 to 16 hours.

Example 5 Induction of Apoptosis in Mast Cells by B. cepacia CytotoxicFactors

Mast, cells were isolated by the method described by Melnikov A. et al.,Mol, Microbiol 36: 1481-1493 (2000). B. cepacia fractionated growthmedium was prepared as in Example 2. Induction of apoptosis in mastcells by B. cepacia cytotoxic factor was determined using confocalmicroscopy, as described in Example 4.

Nontreated mast cells or mast cells, treated overnight with the SUP,HAFT or AAFT fractions of B. cepacia growth medium, fluoresced primarilyred, indicating a lack of apoptotic cell death. Mast cells treatedovernight with the QSFT fraction of B. cepacia growth medium fluorescedmostly green, indicating the apoptotic death of most of the mast cells.

Example 6 Induction of Apoptosis in Macrophages by B. cepacia and M.bovis QSFT Fractions

Macrophage isolation was as in Example 2. Induction of apoptosis inmacrophages by B. cepacia and M. bovis cytotoxic factors was determinedusing the methods of Example 4. Induction of apoptosis of macrophageswas observed when they were treated with the B. cepacia and M. bovisQSFT fractions.

Example 7 Measurement of Caspase Activities (caspase-3 and caspase-9) inthe Cytosolic Extracts of Macrophages Treated with the B. cepacia QSFTFraction

Macrophage isolation was as in Example 2. Macrophages are treatedovernight with the B. cepacia QSFT fraction using the method describedin Example 2. The preparation of macrophage cytoslic extract and thecaspase assays were as described by Zaborina O. et al., Microbiology146: 2521-2530 (2000).

Briefly, determination of caspase-3 activity was performed usingAc-DEVD-pNA (N-acetyl-Asp-Glu-Val-Asp-p-NO₂-aniline) as a substrate.Release of pNA (p-nitroaniline) was determined spectrophotometrically at405 nm from the caspase-3 substrate (200 μm) after 15, 30, 45, 60, 75and 90 min incubation at 37° C. (FIG. 3A) with uninduced macrophagecytosolic extract; cytosolic extract of macrophages incubated, overnightwith the B. cepacia QSFT fraction (10 μg protein); and cytosolic extractof macrophages incubated overnight with the B cepacia QSFT fraction (10μg protein) and added inhibitor (DEVD-CHO). 10 μg of macrophagecytosolic protein was used in each case.

In the caspase-9 assay, release of pNA from 200 μM of the caspase-9substrate Ac-LEHD-pNA (N-acetyl-Leu-Glu-His-Asp-p-NO₂-aniline) wasdetermined, after 15, 30, 45, 60, 75 and 90 min incubation (FIG. 3B),with uninduced macrophage cytosolic extract, cytosolic extract ofmacrophages incubated overnight with the B. cepacia QSFT fraction (10 μgprotein) and cytosolic extract of macrophages incubated overnight withthe B. cepacia QSFT fraction (10 μg protein) plus inhibitor (LEHD-CHO).10 μg of macrophage cytosolic protein was used in each case.

DEVD-CHO and LEHD-CHO respectively block Caspase 3 and Caspase 9activity and are available from Biomol Research Laboratories, PlymouthMeeting, Pa., U.S.A. The activities of both caspase-9 and caspase-3increased when macrophages were treated overnight with the B. cepaciaQSFT fraction (FIGS. 3A and B). These activities remained very low foruntreated macrophages or with inhibitor present, suggesting that theinduction of apoptosis by the QSFT fractions involves caspaseactivation.

Example 8 TUNEL Assay to Measure Nuclear DNA Fragmentation inMacrophages Treated with M. bovis or B. cepacia QSFT Fractions

Fractionated B. cepacia growth medium was obtained using the methoddescribed in Example 2. M. bovis BCG was grown in Middlebrook 7H9 broth(Difco Laboratories, Maryland, U.S.A.) supplemented with 2% glycerol,0.02% TWEEN®80 and ADC (ablumin/dextrose/citrate) (available from DifcoLaboratories, Maryland, U.S.A.). The bacteria were grown for severaldays at 32° C. on a shaker before harvesting. Fractionated M. bovisgrowth medium was obtained using the method described in Example 2.Macrophage isolation was as in Example 2. Induction of apoptosis inmacrophages either untreated or treated by overnight incubation of theSLIP or the HAFT, AAFT or QSFT fractions was measured using confocalmicroscopy by detecting apoptosis-induced nuclear DNA fragmentation withthe ApoAlert DNA fragmentation kit (Clontech Laboratories, Inc., PaloAlto, Calif., U.S.A.). This assay is based on terminaldeoxynuclotidyltransferase (Tdt)—mediated dUTP nick-end labeling(TUNEL), where Tdt catalyzes the incorporation of fluorescein-dUTP atthe free 3′-hydroxyl ends of fragmented DNA in cells undergoingapoptosis. The incorporation of fluorescein-dUTP in the fragmentednuclear DNA generates green fluorescence which is detected by confocalmicroscopy.

Macrophages treated with either the M. bovis or B. cepacia QSFTfractions showed a yellow-green nucleus in the red cytoplasmicbackground, indicating nuclear DNA fragmentation. Little or nofragmentation was observed with untreated macrophages or withmacrophages treated with other column fractions.

Example 9 SDS-PAGE Analysis of Proteins in the Supernatant and the AAFT,HAFT and QSFT Fractions of Growth Media from P. aeruginosa, B. cepaciaand M. bovis

SDS-PAGE separation showed the proteins present in the supernatant andthe AAFT, HAFT and QSFT Fractions of P. aeruginosa, B. cepacia and M.bovis. The QSFT medium traction from mucoid P. aeruginosa strain 8821showed the presence of two bands, a 18 kDa band corresponding to azurinby N-terminal analysis and a 9 kDa band corresponding to cytochromec₅₅₁. The B. cepacia QSFT fraction showed the presence of threepredominant bands of 75 kDa, 20 kDa and 8 kDa. The N-terminal amino acidsequence of 10 amino acids of the 20 kDa band (AHHSVDIQGN), determinedby Edman degradation, showed 80% sequence homology to that of theN-terminal 10 amino acid sequence of P. aeruginosa azurin while theN-terminal amino acid sequence of 10 amino acids of the 8 kDa band(EDPEVLFKNK) showed 100% match with that of P. aeruginosa cytochromec₅₅₁. Thus the QSFT fractions having high cytotoxic activity of both P.aeruginosa and B. cepacia show enrichment with azurin and cytochromec₅₅₁ type of redox proteins. In contrast, the M. bovis QSFT fractionshowed a thick 65 kDa band of bovine serum albumin (BSA), which is aconstituent of the 7H9 medium used for growing M. bovis, as well asseveral bands of greater than 45 kDa molecular mass, but not the 8 kDaor 22 kDa cytochrome c₅₅₁ or azurin type of proteins.

Example 10 Cell Death in Macrophages Treated with Azurin/Cytocbrome c₅₅₁

Purified azurin and cytochrome c₅₅₁ (Sigma Chemicals, St. Louis U.S.A.)were added to macrophages, prepared as in Example 2, and the mixtureincubated for 2 hrs. Azurin and cytochrome c₅₅₁ concentrations were asin FIG. 4. The numbers represent μg protein. Macrophage cell death wasmeasured by the release of the intracellular enzyme lactatedehydrogenase (LDH) using the method of Example 2. Both azurin andcytochrome c₅₅₁ caused macrophage cell death. A combination of azurinand cytochrome c₅₅₁ caused more extensive macrophage cell death. Thebuffer control (buffer) is shown at right. (FIG. 4).

Example 11 Induction of Apoptosis in Macrophages Treated withAzurin/Cytochrome c₅₅₁

Macrophage isolation was as in Example 2. The macrophages were treatedwith azurin/cytochrome c₅₅₁ (50/25 μg) for 4 and 6 hours and thenexamined by confocal microscopy, using the ApoAlert MitochondriaMembrane Sensor kit as in Example 4, to determine the extent ofapoptosis. Macrophages underwent increasing levels of apoptosis withincreasing periods of incubation in presence of azurin/cytochrome c₅₅₁mixture. Control macrophages without treatment (treated withphosphate-buffered saline for 6 hours) did not show apoptosis.

Example 12 Cytotoxicity of an Azurin/Cytochrome c₅₅₁ Mixture or the QSFTFractions Derived from B. cepacia or M. bovis in Macrophages afterPretreatment with Anti-azurin and Anti-cytochrome c₅₅₁ Antibodies

Macrophage isolation was as in Example 2. Macrophages were treated witha purified azurin/cytochrome c₅₅₁ mixture (50/25 μg), or the B. cepaciaor M. bovis QSFT fractions in the presence and absence of a mixture ofanti-azurin and anti-cytochrome c₅₅₁ antibodies prepared in rabbits.Antibodies were mixed in a ratio of 1:1 and the mixed antibody (1, 2, 3,or 4 mg) was used for treatment of macrophages.

The extent of macrophage cell death was determined by release of the LDHas in Example 2. FIG. 5 shows a reduction of cytotoxicity towardsmacrophages treated with an azurin/cytochrome c₅₅₁ mixture (A+C), or theQSFT fraction derived from B. cepacia (Bc-QSFT), when anti-azurin andanti-cytochrome c₅₅₁ antibodies are present. This reduction was notobserved with the QSFT fraction from M. bovis (Mb-QSFT).

Hence, when an azurin/cytochrome c₅₅; mixture or the B. cepacia QSFTfraction was treated with a mixture of anti-azurin and anti-cytochromec₅₅₁ antibodies, and then assayed for macrophage cytotoxicity, thecytotoxicity was greatly diminished. In contrast, when the M. bovis QSFTfraction, which was previously shown by SDS-PAGE gel to lack azurin andcytochrome c₅₅₁ bands (Example 9), was pretreated withanti-azurin/anti-cytochrome c₅₅₁ antibodies and then assayed forcytotoxicity, very little reduction in cytotoxicity was observed.

Example 13 Induction of Apoptosis in Tumor Cell Lines by the B. cepaciaQSFT Fraction and by Azurin/Cytochrome c₅₅₁ as Measured by ConfocalMicroscopy

H460 lung carcinoma, PA-1 ovarian cancer, NCF breast cancer, HT-29 coloncancer and HT-1080 leukemia cell lines were obtained from the AmericanType Culture Collection (Manassas, Va., U.S.A.). MDD7 and MN1 breastcancer cell lines were obtained from Andrei Gudkov, Ph.D., ClevelandClinic Foundation (Cleveland, Ohio U.S.A.). UISO-BCA-9 breast cancer andUSIO-MEL-1, MEL-2. MEL-6 and MEL-29 melanoma cell lines were developedand maintained as described in Rauth, S et al. In vitro Cellular andDevelopmental Biology, 30a(2): 79-84 (1994) and Rauth, S et al.,Anticancer Research, 14(6): 2457-2463 (1994). Approximately 1×10⁵ ofeach cell type were cultured overnight in a 0.15 mm thick dTC3 dish(Bioptech, Butler, Pa. U.S.A.) in the presence of the B. cepacia QSFTfraction (5 μg protein) or a azurin/cytochrome c₅₅₁ mixture (50/25 μg).The cells were subsequently examined by confocal microscopy, as inExample 4, to determine the extent of apoptosis. Both the B. cepaciaQSFT fraction the and azurin/cytochrome c₅₅₁ mixture induced extensiveapoptosis in H460 lung carcinoma. HT-29 colon cancer, HT-1080 leukemia,PA-1 ovarian cancer, MDD7, NCF and MN1 breast cancer, and USIO-MEL-1,MEL-2, MEL-6 and MEL-29 melanoma cells after overnight incubation. Ineach case, cells not treated with cytotoxic factor (phosphate-bufferedsaline added) did not show extensive apoptosis.

Example 14 Induction of Apoptosis in USIO-Mel-6 Melanoma Cell Line bythe M. bovis QSFT Fraction as Measured by TUNEL Assay

USIO-Mel-6 melanoma cells were prepared as described in Rauth, S et al.,Anticancer Research, 14(6): 2457-2463 (1994). M. bovis QSFT fraction wasprepared as in Example 8. The melanoma cells treated with M. bovis QSFTfraction (5 μg protein) and untreated control cells were incubated for12 hours. Induction of apoptosis was measured using the TUNEL assay todetect apoptosis-induced nuclear DMA fragmentation as in Example 8.Melanoma cells treated with the M. bovis QSFT fraction showed ayellow-green nucleus in the red cytoplasmic background, indicatingnuclear DNA fragmentation. Little or no fragmentation was observed withuntreated melanoma cells.

Example 15 Reduction of Growth of Melanoma Tumor Cells (USIO-Mel-2) inNude Mice after Treatment with Azurin/Cytochrome c₅₅₁

Approximately 10⁶ USIO-Mel-2 cells were injected subcutaneously in nudemice (available from Frederick Cancer Research and Development Center,Frederick, Maryland U.S.A.). Small tumors developed after approximatelythree weeks. The mice then received once weekly intraperitonealinjections of a known anti-melanoma drug, DTIC [5(3,3′-N,N-dimethyltriazen-1-yl)-imidazole-4-carboxamide] (7.5 μg) (see Ahlmais et al.,Cancer 63: 224-7 (1989)) or three weekly intraperitoneal injections of ahigh (150 μg azurin/75 μg cytochrome c₅₅₁), low (10 μg azurin/5 μgcytochrome c₅₅₁) dose of azurin/cytochrome c₅₅₁ mixture or control(citrate buffer) for 4 weeks. The tumor volume was determined atintervals in the control, DTIC-treated, and high and low doseazurin/cytochrome c₅₅₁-treated mice.

The increases in tumor size in control, DTIC-treated andazurin/cytochrome-treated nude mice are shown in FIG. 6 and the weightgain/loss data in such mice are shown in FIG. 7. Post-injection of ahigh dosage of 150 μg azurin/75 μg cytochrome-c₅₅₁ produced delayedgrowth and a shrinkage of the tumor size comparable of DTIC. FIG. 7shows that the injection of either DTIC or azurin/cytochrome c₅₅₁mixture did not affect the weight gain of the mice. All mice gainedweight during the experimental period.

Example 16 Effect of Post Injection of Azurin and M. bovis QSFT Fractionin Nude Mice on Tumor Size after Injection of Melanoma Tumor Cells(Mel-6)

Approximately 10⁶ USIO-Mel-6 cells were injected subcutaneously in 3nude mice (available from Frederick Cancer Research and DevelopmentCenter, Frederick, Md. U.S.A.). Small tumors developed afterapproximately three weeks. One mouse was then injected intraperitoneallywith phosphate-buffered saline (control), one mouse was injected with M.bovis QSFT fraction (5 μg protein) and one mouse was injected with amixture of M. bovis QSFT fraction (5 μg protein) and Azurin (50 μg). TheM. bovis QSFT fraction was prepared as in Example 8. The sizes (tumorvolume) of the tumors in control, M. bovis QSFT fraction treated and M.bovis QSFT fraction/Azurin treated mice were determined over a period of30 days. These data are shown in FIG. 8. Both the treated nice showeddecreased tumor growth compared to the control mouse.

What is claimed is:
 1. A pharmaceutical composition comprising:cytotoxic factors comprising an azurin and a cytochrome c₅₅₁; and apharmaceutically-acceptable carrier; wherein the dosage form is selectedfrom the group consisting of: a tablet, a pill, a dragee and a gel; andwherein the cytotoxic factors are present in a therapeutically effectiveamount to cause the death of cancer cells in the patient, or inhibit thegrowth of cancer cells in the patient.
 2. The pharmaceutical compositionof claim 1, wherein the azurin is from Pseudomonas aeruginosa.
 3. Thepharmaceutical composition of claim 1, wherein the cancer cells areselected from the group consisting of: melanoma cells, leukemia cells,breast cancer cells, ovarian cancer cells, lung cancer cells,mesenchymal cancer cells, colon cancer cells, and aerodigestive tractcancer cells.
 4. The pharmaceutical composition of claim 1 wherein thedosage form is suitable for administering to the patient by oral,buccal, sublingual, rectal, vaginal, transurethral, nasal, topical,percutaneous, transdermal, intramuscular, or subcutaneousadministration.
 5. A method of making a dosage form for administrationto a patient with cancer comprising combining an azurin and a cytochromec₅₅₁ in an admixture with a pharmaceutically acceptable carrier; andmaking the admixture into a dosage form selected from the groupconsisting of a tablet, a pill, a dragee, a capsule and a gel, whereinthe cytotoxic factors are present in the dosage form in an amount tocause to cause the death of cancer cells in a patient, or inhibit thegrowth of cancer cells in a patient.
 6. The method of claim 5 whereinthe dosage form is suitable for administering to a patient by oral,buccal, sublingual, rectal, vaginal, transurethral, nasal, topical,percutaneous, transdermal, intramuscular, or subcutaneousadministration.